University of Groningen

Prednisolone has a positive effect on the kidney but not on the liver of brain dead ratsRebolledo Acevedo, Rolando; Liu, Bo; Akhtar, Mohammed Z.; Ottens, Petra J.; Zhang, Jian-ning; Ploeg, Rutger J.; Leuvenink, Henri G. D.Published in:Journal of translational medicine

DOI:10.1186/1479-5876-12-111

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Citation for published version (APA):Rebolledo Acevedo, R., Liu, B., Akhtar, M. Z., Ottens, P. J., Zhang, J., Ploeg, R. J., & Leuvenink, H. G. D.(2014). Prednisolone has a positive effect on the kidney but not on the liver of brain dead rats: a potencialrole in complement activation. Journal of translational medicine, 12, [111]. https://doi.org/10.1186/1479-5876-12-111

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Rebolledo et al. Journal of Translational Medicine 2014, 12:111http://www.translational-medicine.com/content/12/1/111

RESEARCH Open Access

Prednisolone has a positive effect on the kidneybut not on the liver of brain dead rats: a potencialrole in complement activationRolando Rebolledo1,2*†, Bo Liu1,3†, Mohammed Z Akhtar4, Petra J Ottens1, Jian-ning Zhang3,Rutger J Ploeg1,4 and Henri GD Leuvenink1

Abstract

Background: Contradictory evidence has been published on the effects of steroid treatments on the outcomes ofkidney and liver transplantation from brain dead (BD) donors. Our study aimed to evaluate this disparity byinvestigating the effect of prednisolone administration on BD rats.

Methods: BD induction was performed in ventilated rats by inflating a Fogarty catheter placed in the epidural space.Prednisolone (22.5 mg/kg) was administered 30 min prior to BD induction. After four hours of determination of BD:serum, kidney and liver tissues samples were collected and stored. RT-qPCR, routine biochemistry andimmunohistochemistry were performed.

Results: Prednisolone treatment reduced circulating IL-6 and creatinine plasma levels but not serum AST, ALT orLDH. Polymorphonuclear influx assessed by histology, and inflammatory gene expression were reduced in the kidneyand liver. However, complement component 3 (C3) expression was decreased in kidney but not in liver. Geneexpression of HSP-70, a cytoprotective protein, was down-regulated in the liver after treatment.

Conclusions: This study shows that prednisolone decreases inflammation and improves renal function, whilst notreducing liver injury. The persistence of complement activation and the negative effect on protective cellularmechanisms in the liver may explain the disparity between the effects of prednisolone on the kidney and liver of BDrats. The difference in the molecular and cellular responses to prednisolone administration may explain thecontradictory evidence of the effects of prednisolone on different organ types from brain dead organ donors.

Keywords: Brain death, Organ donation, Steroids, Prednisolone, Kidney transplantation, Liver transplantation

BackgroundThe shortage of organs for transplantation remains one ofthe most important issues facing the transplant commu-nity today. Increasingly, organs from brain dead extendedcriteria donors (ECD) and donors after circulatory arrest(DCD) are being used to address the organ deficit. Theshort and long term outcomes of allografts obtained fromthese donors are inferior when compared to living donors

*Correspondence: r.a.rebolledo@umcg.nl†Equal contributors1Department of Surgery, University Medical Center Groningen, Hanzeplein 1,9713 GZ Groningen, The Netherlands2Department of Surgery, Faculty of Medicine, University of Chile,Independencia 1027, 8380453 Santiago, ChileFull list of author information is available at the end of the article

[1,2]. In the case of organs obtained from donors afterbrain death (DBD) it has been shown that HLA mis-matched living donors have better outcomes even whencold ischemia times are taken into consideration [3]. Theexplanation for this lies in the process of brain death (BD)itself resulting in a non-physiological environment culmi-nating in significant organ injury prior to organ procure-ment. The procurement, preservation and reperfusionphases of transplantation result in significant additionalinjury to the allograft rendering it susceptible short andlong term dysfunction [4-6].

BD causes complex disturbances of normal homeo-static systems resulting in hemodynamic instability [7-10]hormonal impairment [11-13] and inflammation [14-17].

© 2014 Rebolledo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedicationwaiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwisestated.

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BD results in significant cerebral ischemia and intracra-nial hypertension resulting in parasympathetic activityfollowed by a severe vasoconstriction. This is due toan overriding sympathetic response and termed the“catecholamine storm” which is part of the Cushing reflex;a physiological response to maintain cerebral perfusion. Aprogressive paralysis of the spinal cord occurs, this causethe loss of vasomotor tone and leads to hemodynamicinstability which characterizes this period [18-20].

Pituitary function is also affected following BD asadrenocorticotrophic hormone (ACTH) secretion isaltered resulting in a transient rise in cortisol levels,which then diminishes progressing below baseline levels.In addition other hormones including T4/T3, anti-diuretichormone (ADH) and insulin are reduced as a consequenceof BD [21].

BD results in a heightened systemic inflammatorystate as illustrated by the influx of polymorphonuclearneutrophils (PMNs) into kidney and liver tissues [15].Systemic production of circulating cytokines includ-ing interleukin-6 (IL-6), interleukin-10 (IL-10), TumorNecrosis Factor-alpha (TNF-α), Transforming GrownFactor-beta (TGF-β) and Monocyte chemotactic protein1 (MCP-1) are thought to orchestrate this response. Thetrigger for this inflammatory process is not well under-stood, however recent evidence suggests there could bea role for cerebral cytokines that cross the blood-brainbarrier, in addition to complement activation [22,23] andintestinal bacterial translocation [24,25].

A number of important considerations have beendescribed for the ICU management of BD organ donors.There is general consensus about the importance of main-taining hemodynamic stability, but hormonal or anti-inflammatory treatments remains controversial despitepromising experimental evidence [21]. The adminis-tration of glucocorticoids to donors has been used ashormonal replacement therapy with a varied degree ofsuccess [26-31].

In BD glucocorticoid administration could have sev-eral beneficial effects including anti-inflammatoryproperties and the ability to augment chrommaffincells production of endogenous epinephrine [32]. Theanti-inflammatory effects of steroids result from thepleiotropic interaction with the glucocorticoid receptor.The cortisol-glucocorticoid receptor complex can actthrough genomic and non-genomic downstream sig-nalling pathways within the cell. This involves thedissociation of heat-shock proteins and the interactionwith membrane-associated receptors, second messengersand activation of transcription factors such as Nuclearfactor kappa-light-chain-enhancer of activated B cells(NF-κB) [33].

We hypothesize that prednisolone pre-treatment of BDrats could reduce the inflammatory response and improve

the quality of kidney and liver allografts. The aim ofthis study is to assess the potential benefit of the pred-nisolone pre-treatment in the liver and kidney of BDrats.

MethodsAnimalsMale adult Fisher F344 rats (250 - 300 g) were usedin all experiments. All animals received care in com-pliance with the guidelines of the local animal ethicscommittee according to Experiments on Animals Act(1996) issued by the Ministry of Public Health, Welfareand Sports of the Netherlands. Brain death (BD) wasinduced as follows: animals were anesthetized usingisoflurane with O2. A cannula was inserted in thefemoral artery and vein for continuous mean arterial pres-sure (MAP) monitoring and fluid administration. Ani-mals were intubated via a tracheostomy and ventilatedthroughout the experiment. A no. 4 Fogarty catheter(Edwards Lifesciences Co, Irvine, CA) was placed in theepidural space through a frontolateral burr hole, andslowly inflated (0.16 ml/min) with saline using a syringepump (Terufusion, Termo Co, Tokyo, Japan). Inflationof the balloon was terminated once the MAP beganto improve after a characteristic period of hypotension,reflecting the autonomic storm. BD was confirmed bythe absence of corneal and pupillary reflexes and a pos-itive apnea test. Following confirmation of BD, anes-thesia was terminated but ventilation continued. MAPswere maintained above 80mmHg using Hydroxyethylstarch (HAES) 10% (Fresenius Kabi AG, Bad Homburg,Germany) with a maximum rate of 1 ml/hr. If HAESwas insufficient to maintain the MAP, noradrenaline (NA)0.01 mg/ml was administered. A homeothermic blan-ket control system was used throughout the BD main-tenance period. Four hours after determination of BD,rats were heparinized with 500 IU heparin. A laparo-tomy was subsequently performed and blood collectedfrom the aorta. Organs were flushed with 0.9% saline andsnap frozen in liquid nitrogen and collected plasma storedat −80°C.

Rats were randomly assigned to each group. The ani-mal number was calculated using the method of RussLenth [34], with a meaningful difference of 50%, a vari-ability (sigma) of 0.3 and a power of 0.9. Sham-operatedrats served as controls and were ventilated for half anhour under anesthesia before termination. This was inaccordance with the requirement of our local AnimalWelfare Committee guidance for the use of sham controlsin experiments. Prednisolone or saline was administeredintravenously 30 minutes before the start of BD induc-tion. Prednisolone dosage was chosen based on previousexperiments and to give the best hemodynamic stability(22.5 mg/kg).

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The following experimental groups were established:

• Sham-operated animals receiving saline (n = 8).• Sham-operated animals receiving Prednisolone

(n = 8).• BD animals receiving saline (n = 8).• BD animals receiving Prednisolone (n = 8).

Plasma determinationsPlasma creatinine was determined in the biochemistry labof the University Medical Center Groningen. The level ofIL-6 in the plasma was determined by a rat enzyme-linkedimmunosorbent assay (IL-6 ELISA) kit (R&D SystemsEurope Ltd. Abingdon, Oxon OX14 3NB, UK), accord-ing to the manufacturer instructions. All samples wereanalyzed in duplicate and read at 450 nm.

RNA isolation and cDNA synthesisTotal RNA was isolated from whole kidneys andliver sections by using TRIzol (Life Technologies,Gaithersburg, MD). RNA samples were verified forabsence of genomic DNA contamination by performingRT-PCR reactions in which the addition of reverse tran-scriptase was omitted, using Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) primers. For cDNA synthesis,1 μl T11VN Oligo-dT (0.5 μg/μl) and 1 μg mRNA wereincubated for 10 min at 70°C and cooled directly afterthat. cDNA was synthesized by adding a mixture con-taining 0.5 μl RnaseOUT Ribonuclease inhibitor (Invit-rogen, Carlsbad, USA), 0.5 μl RNase water (Promega),4 μl 5 x first strand buffer (Invitrogen), 2 μl DTT (Invitro-gen), 1 μl dNTPO’s and 1 μl M-MLV reverse transcriptase(Invitrogen, 200U). The mixture was held at 37°C for 50min. Next, reverse-transcriptase was inactivated by incu-bating the mixture for 15 min at 70°C. Samples werestored at −20°C.

Real-Time PCRFragments of several genes were amplified with the primersets outlined in Table 1. Pooled cDNA obtained from

brain-dead rats were used as internal references. Geneexpression was normalized with the mean of β-actinmRNA content. Real-Time PCR was carried out in reac-tion volumes of 15 μl containing 10 μl of SYBR Greenmastermix (Applied biosystems, Foster City, USA), 0.4 μlof each primer (50 μM), 4.2 μl of nuclease free water and10 ng of cDNA. All samples were analyzed in triplicate.Thermal cycling was performed on the Taqman AppliedBiosystems 7900HT Real Time PCR System with a hotstart for 2 min at 50°C followed by 10 min 95°C. Secondstage was started with 15 s at 95°C (denaturation step)and 60 s at 60°C (annealing step and DNA synthesis). Thelatter stage was repeated 40 times. Stage 3 was includedto detect formation of primer dimers (melting curve) andbegins with 15 s at 95°C followed by 60 s at 60°C and 15 sat 95°C. Primers were designed with Primer Express soft-ware (Applied Biosystems) and primer efficiencies weretested by a standard curve for the primer pair resultingfrom the amplification of serially diluted cDNA samples(10 ng, 5 ng, 2.5 ng, 1.25 ng and 0.625 ng) obtainedfrom brain-dead rats. PCR efficiency were found to be1.8 < ε < 2.0. Real-time PCR products were checked forproduct specificity on a 1.5% agarose gel. Results wereexpressed as 2-�� CT (CT: Threshold Cycle).

ImmunohistochemistryTo detect polymorphonuclear cells in kidney and liver,immunohistochemistry was performed on 5-μm tis-sue cryosections. Sections were fixated for 10 minusing acetone. Next, sections were stained with HIS-48mAb (supernatant, two times diluted) using an indirectimmunoperoxidase technique. Endogenous peroxidasewas blocked using H2O2 0.01% in phosphate-bufferedsaline for 30 mins. After thorough washing, sections wereincubated with horseradish peroxidase-conjugated rabbitanti-mouse IgG as a secondary antibody for 30 mins,followed by goat anti-rabbit IgG as a tertiary antibodyfor 30 mins (both from Dako, Glostrup, Denmark). Thereaction was developed using 9-amino-ethylcarbazole as

Table 1 Primer sequences used for Real-Time PCR

Gene Primers Amplicon size (bp)

TNF-α 5’-GGCTGCCTTGGTTCAGATGT-3’ 5’-CAGGTGGGAGCAACCTACAGTT-3’ 79

IL-1β 5’- CAGCAATGGTCGGGACATAGTT-3’ 5’-GCATTAGGAATAGTGCAGCCATCT-3’ 75

IL-6 5’-CCAACTTCCAATGCTCTCCTAATG-3’ 5’-TTCAAGTGCTTTCAAGAGTTGGAT-3’ 89

C3 5’-CAGCCTGAATGAACGACTAGACA-3’ 5’-TCAAAATCATCCGACAGCTCTATC-3’ .96

MCP-1 5’-CTTTGAATGTGAACTTGACCCATAA-3’ 5’-ACAGAAGTGCTTGAGGTGGTTGT -3’ 78

HO-1 5’-CTCGCATGAACACTCTGGAGAT-3’ 5’-GCAGGAAGGCGGTCTTAGC-3’ 74

HSP-70 5’-GGTTGCATGTTCTTTGCGTTTA-3’ 5’-GGTGGCAGTGCTGAGGTGTT-3’ 80

BAX 5’-GCGTGGTTGCCCTCTTCTAC-3’ 5’-TGATCAGCTCGGGCACTTTAGT-3’ 74

BCL-2 5’-CTGGGATGCCTTTGTGGAA-3’ 5’-TCAGAGACAGCCAGGAGAAATCA-3’ 70

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chromogen and H2O2 as substrate. Sections were coun-terstained using Mayer hematoxylin solution (Merck,Darmstadt, Germany). Negative antibody controls wereperformed. Localization of immunohistochemical stain-ing was assessed by light microscopy. For each tissuesection, positive cells per field were counted in 10 micro-scopic fields of the tissue at 40x magnification. Resultswere presented as number of positive cells per glomerulusin the kidney and number of positive cells per field in theliver.

Statistical analysisStatistical analyses were performed using Prism 5.0.GraphPad. The two-way ANOVA test was performed, fol-lowed by the Bonferroni post-test. Specific analysis for theuse of HAES and NA between groups was evaluated usingthe Mann-Whitney test. The significance level was set ata p value of < 0.05. Results are presented as mean ± SD(standard deviation).

ResultsInduction of BD showed a consistent and uniform patternin alterations of the MAP, with a mean time to declarationof BD of 31.6 ± 2.73 minutes after commencing ballooninflation. All 32 animals were kept with a MAP above80 mmHg during the experiment (Figure 1), after the con-firmation of BD. In saline treated group, infusion of HAES10% (2.7 ml ± 1.1) was needed to maintain MAP whereasin the prednisolone treated group significantly less wasrequired (1.3 ml ± 1.3 HAES 10%). Administration of NA

was comparable between both groups (saline: 0.11 ± 0.12mg, prednisolone: 0.05 ± 0.09 mg).

Plasma creatinine levels was significantly differentbetween sham and BD animals treated with saline solu-tion. Creatinine plasma levels in BD animals treated withprednisolone (50.38 ± 6.90) was significantly lower com-pared to BD rats pre-treated with saline (86.63 ± 18.37)(Figure 2).

Assessing cellular liver injury, aspartate aminotrans-ferase (AST), alanine aminotransferase (ALT) and lactatedehydrogenase (LDH) plasma levels were significantlyincreased after BD in comparison to sham animals. Nosignificant differences were found in AST, ALT or LDHplasma levels between prednisolone or saline treated BDrats (Figure 2).

Our results demonstrated a significant decrease in IL-6levels in BD animals treated with prednisolone comparedto BD animals treated with saline (Figure 2).

Tissue cryosections were stained with HIS-48 mAb toidentify PMNs. The number of positive cells per glomeru-lus in the kidney was 0.67 ± 0.4 in sham-operated controlsbut was elevated to 1.42 ± 0.4 positive cells in the BD+ saline group. Treatment with prednisolone reduced thePMN influx to 0.86 ± 0.4 positive cells, reaching statisticalsignificance compared to the BD saline group (Figure 3).

In the case of the liver, the number of positive cells permicroscopic field was 5.85 ± 2.0 in sham-operated con-trols but was elevated to 13.41 ± 4.0 positive cells in theBD + saline group. Treatment with prednisolone signifi-cantly diminished the PMN influx to 6.97 ± 2.5 positivecells (Figure 3).

Figure 1 Blood pressure registry. The record started with the brain death induction, we considered the time “0” as the end of brain deathinduction and the starting of BD period. As a black-continuous line the mean in each minute of blood pressure for the prednisolone brain deathgroup. As a grey-continuous line the mean in each minute for the saline brain death group.

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Figure 2 Plasmatic levels of kidney function markers and liver injury markers. Creatinine (Sham operated + Saline: 36 ± 9.24 μM; Shamoperated + prednisolone treatment: 36.88 ± 4.29 μM; BD + Saline 86.63 ± 18.37 μM; BD + prednisolone treatment: 50.38 ± 6.91 μM), AST (Shamoperated + Saline: 71.50 ± 14.19 U/l; Sham operated + prednisolone treatment: 72.88 ± 11.66 U/l; BD + Saline: 138.0 ± 72.76 U/l; BD + prednisolonetreatment: 146.3 ± 35.07 U/l), ALT (Sham operated + Saline: 54.38 ± 13.97 U/l; Sham operated + prednisolone treatment: 45.88 ± 15.82 U/l;BD + Saline: 106.3 ± 58.38 U/l; BD + prednisolone treatment: 87.50 ± 18.33 U/l), LDH (Sham + Saline: 157.1 ± 41.54 U/l; Sham + prednisolonetreatment: 172.4 ± 104.2 U/l; BD + Saline: 342.8 ± 228.0 U/l; BD + prednisolone treatment: 270 ± 152.9 U/l) and IL-6 (Sham operated + Saline:15.63 ± 29.69 pg/ml; Sham operated + prednisolone: undetectable; BD + Saline: 2.07 × 105 ± 9.59 × 104 pg/ml; BD + prednisolone treatment:1.18 × 103 ± 2.69 × 103 pg/ml).

Figure 3 PMN infiltration quantification and stanning. Kidneys from A) Sham operated + Saline, B) Sham operated + prednisolone treatment,C) BD + Saline, D) BD + prednisolone treatment rats. PMN infiltration in livers from E) Sham operated + Saline, F) Sham operated + prednisolonetreatment, G) BD + Saline, H) BD + prednisolone treatment rats, 40x magnification. I) Quantification of PMN infiltration in kidney and J) liver.

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qRT-PCR evaluation of gene expression revealed signif-icantly upregulated IL-6, Interleukin 1 beta (IL-1β) andmonocyte chemoattractant protein-1 (MCP-1) in kidneysas a consequence of BD compared to sham controls. Rel-ative expression of IL-6, IL-1β , MCP-1 was significantlyreduced in the prednisolone treated groups in the kidneysamples (Figure 4).

In liver, relative expression of IL-6, IL-1β and MCP-1 was significantly increased following BD. Prednisolonetreatment significantly reduced IL-6, IL-1β and MCP-1compared to the BD control group (Figure 4).

TNF-α is a monocyte derived cytotoxin implicated inmediating apoptosis. In the kidney the relative expres-sion of TNF-α increased but not to a significant levelfollowing BD. TNF-α was significantly down-regulatedfollowing prednisolone pre-treatment. However, in theliver the relative expression of TNF-α was significantlydown-regulated following BD and further down-regulatedfollowing prednisolone treatment in the BD group(Figure 4).

In the kidney C3 expression was increased follow-ing BD and attenuated following prednisolone treatment.

While in the liver, C3 expression increased followingBD and further increased due to prednisolone treatment(Figure 5).

To assess the effect on apoptotic pathways we stud-ied the B-cell lymphoma 2 (Bcl2)/Bcl-2 associatedX protein (BAX) ratio. In the kidney this ratio wasincreased following BD but decreased in the liver.Prednisolone treatment did not significantly mod-ify this ratio under any experimental condition(Figure 5).

We studied the effects of the interventions on therelative expression of two cytoprotective genes. Therelative expression of Heme oxygenase 1 (HO-1) inthe kidney was up-regulated following BD and down-regulated following pre-treatment with prednisolone.In the liver the HO-1 expression was significantlyup-regulated following prednisolone treatment. Therelative HSP-70 expression in the kidney was notaffected by BD nor prednisolone treatment. How-ever, the relative expression in the liver was signifi-cantly down-regulated due to prednisolone pre-treatment(Figure 5).

Figure 4 Relative expression of inflammatory genes. A) Kidney samples. IL-6 (Sham operated + Saline: 0.08 ± 0.09; Sham operated +prednisolone treatment: 0.05 ± 0.05; BD + Saline: 6.04 ± 3.79; BD + prednisolone treatment: 0.34 ± 0.59), IL-1β (Sham operated + Saline: 0.89 ±0.22; Sham operated + prednisolone treatment: 0.27 ± 0.11; BD + Saline: 1.95 ± 0.69; BD + prednisolone treatment: 0.40 ± 0.30), TNF-α (Shamoperated + Saline: 0.69 ± 0.34; Sham operated + prednisolone treatment: 0.20 ± 0.10; BD + Saline: 0.99 ± 0.49; BD + prednisolone treatment: 0.20 ±0.16), MCP-1 (Sham operated + Saline: 0.46 ± 0.13; Sham operated + prednisolone treatment: 0.13 ± 0.07; BD + Saline 4.46 ± 1.02; BD +prednisolone treatment: 0.39 ± 0.44). B) Liver samples. IL-6 (Sham operated + Saline: 0.11 ± 0.05; Sham operated + prednisolone treatment: 0.04 ±0.03; BD + Saline: 2.98 ± 0.84; BD + prednisolone treatment: 0.60 ± 0.45), IL-1β (Sham operated + Saline: 1.09 ± 0.42; Sham operated + prednisolonetreatment: 0.50 ± 0.20; BD + Saline: 1.91 ± 0.27; BD + prednisolone treatment: 0.61 ± 0.26), TNF-α (Sham operated + Saline: 7.91 ± 2.13; Shamoperated + prednisolone treatment: 4.51 ± 1.57; BD + Saline: 4.04 ± 0.66; BD + prednisolone treatment: 1.3 ± 0.71), MCP-1 (Sham operated + Saline:0.71 ± 0.17; Sham operated + prednisolone treatment: 0.32 ± 0.09; BD + Saline: 1.06 ± 0.23; BD + prednisolone treatment: 0.36 ± 0.24).

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Figure 5 Relative expression of C3, HO-1 and HSP-70. Ratio of Bcl2/BAX expression. A) Kidney samples. C3 (Sham operated + Saline: 0.18 ± 0.04;Sham operated + prednisolone treatment: 0.16 ± 0.02; BD + Saline: 17.27 ± 10.74; BD + prednisolone treatment: 1.47 ± 0.26), HO-1 (Sham operated +Saline: 0.03 ± 0.01; Sham operated + prednisolone treatment: 0.04 ± 0.01; BD + Saline: 3.05 ± 2.06; BD + prednisolone treatment: 0.42 ± 0.23),HSP-70 (Sham operated + Saline: 0.06 ± 0.09; Sham operated + prednisolone treatment: 0.03 ± 0.07; BD + Saline: 0.03 ± 0.02; BD + prednisolonetreatment: 0.03 ± 0.01), Ratio Bcl2/BAX (Sham operated + Saline: 0.37 ± 0.15; Sham operated + prednisolone treatment: 0.38 ± 0.15; BD + Saline:0.69 ± 0.19; BD + prednisolone treatment: 0.69 ± 0.28). B) Liver samples. C3 (Sham operated + Saline: 546.9 ± 109.4; Sham operated + prednisolonetreatment: 534.2 ± 92.18; BD + Saline: 687.4 ± 96.13; BD + prednisolone treatment: 952.2 ± 125.3), HO-1 (Sham operated + Saline: 0.47 ± 0.11; Shamoperated + prednisolone treatment: 0.45 ± 0.13; BD + Saline: 5.78 ± 2.65; BD + prednisolone treatment: 12.47 ± 4.96), HSP-70 (Sham operated +Saline: 0.05 ± 0.09; Sham operated + prednisolone treatment: 0.05 ± 0.11; BD + Saline: 0.03 ± 0.04; BD + prednisolone treatment: 0.003 ± 0.005),Ratio Bcl2/BAX (Sham operated + Saline: 0.18 ± 0.03; Sham operated + prednisolone treatment: 0.18 ± 0.01; BD + Saline: 0.05 ± 0.01; BD +prednisolone treatment: 0.05 ± 0.01).

DiscussionDisparity exists in the literature with regards to the bene-ficial effects of prednisolone administration to the donoron the outcomes of solid organ transplantation. Multi-ple large randomized control trials (RCT) have evaluatedthe effects on steroid administration on the outcomes oflung, kidney and liver transplant [35-37]. In a RCT, Bonseret al. evaluated the effects of 1g of methylprednisoloneadministration to BD organ donors 7 hours prior to lungexplantation, demonstrating no effect on increasing lungyields [29]. Others in the realms of kidney transplan-tation have also failed to detect a significant improve-ment in the kidney survival at three months when 5g ofmethylprednisolone is administered to donors 2-4 hoursprior to explantation. The only clinical trial to suggest apositive outcome was performed by Kotsch et al. demon-strating a significant effect on down-regulation of pro-inflammatory cytokines and reduced incidence of acuterejection [28]. In contrast, an Austrian study failed toidentify a benefit of 1g of methylprednisolone administra-tion 6 hours prior to liver recovery [38].

Despite the heterogeneity of outcomes of these tri-als, steroid administration has, in many countries, beenincorporated into donor management protocols. A reviewby Bugge et al. suggests the routine administration ofmethylprednisolone in all DBDs [39]. Indeed in the UKmethylprednisolone is being trialed as part of a nationaldonor optimization care-bundle pathway. Overall there isa clear need to establish the effects of steroid treatment inthe donor and evaluate the potential differential effects oftreatment on different organ systems.

This study was designed as a proof of concept studyto evaluate the differential effects of prednisolone admin-istration to brain dead rats. The results of our studysuggest that the effect of steroid therapy in the donorwill have differential effects on different organ sys-tems. Our study confirms that steroid administration canimprove the hemodynamic stability of the BD rodent.We hypothesize this effect occurs through facilitating theretention of sodium and water, and increasing the releaseof endogenous epinephrine [40]. Outside of this study,donor hemodynamic stability has been shown to reduce

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pre-procurement injury to allografts and improve shortand long-term outcomes [41,42].

Our study has also shown that steroid administrationdoes have an anti-inflammatory effect as illustrated by thereduction of circulating IL-6. IL-6 has been shown to besignificantly up-regulated following BD and is hypothe-sized to be a key cytokine mediating the inflammatorycellular response to BD through an NFκB mediated path-way with the downstream effectors being selectin, MCP-1and TNF-α [43]. Indeed authors speculate that the linkbetween cerebral injury and peripheral organ injury lies inan IL-6 mediated inflammatory response.

In our study prednisolone had different and disparenteffects on kidney and liver allografts. In the kidney wedemonstrated a significant improvement on kidney func-tion, as assessed by serum creatinine and a reduction ininfiltrating PMNs. This was accompanied by a reductionin expression of IL-6, MCP1 and IL-1β , we demonstrateda reduction of TNF-α expression following methylpred-nisolone administration although this did not reach sig-nificance. We were able to demonstrate a reduction inC3 expression, having previously demonstrated the poten-tial detrimental effects of complement activation in theBD donor [22,44,45]. We believe these anti-inflammatoryeffects, in conjunction with the hemodynamic stabilityconferred by prednisolone administration reduces theinjury to the kidney and result in the improved kidneyfunction.

Our study also demonstrated a reduction in HO-1expression in the kidney following methylprednisolonepretreatment of BD rats. As has been shown by Terry et al.[46] HO-1 is up-regulated by inflammatory cytokineslike IL-1α via protein kinase C and phospholipase A2in endothelial cells. We hypothesize that prednisoloneis down-regulating HO-1 in the kidney by reducing theexpression of inflammatory cytokines. In contrast to thekidney, in the liver HO-1 expression was increased fol-lowing prednisolone pretreatment of the brain dead rat,even when the expression of inflammatory genes wasdecreased. We think that these results could be an indica-tion of the cellular stress during the brain death period.

In the liver we failed to detect a significant effect ofprednisolone pre-treatment of the BD rat to reduce hepa-tocyte injury as assessed by AST and ALT, we did howeverdemonstrate a significant effect on reducing PMN infil-tration and IL-6, MCP-1 and IL-1β expression. We foundthat TNF-α expression was reduced in the liver followingBD in comparison to sham operated animals. This effectmight be related with the surgical stress in both groupsand could be explain by a shorter timeframe between thesurgical procedure and the organ retrieval in sham oper-ated animals. However, TNF-α expression was furtherreduced in the prednisolone pretreated BD rats compareto the saline pretreated BD animals. TNF-α affects cell

cycle and apoptotic pathways through NFκB and MAPkinase mediated pathways. Our previous work has sug-gested TNF-α expression is increased following BD in theliver and systemically. We have previously been able toreduce systemic TNF-α release by stimulating the vagusnerve in a rodent model of brain death [47]. ConcurrentlyKotch et al. demonstrated pre-treatment of human donorswith methylprednisolone could reduce TNF-α expression,supporting our findings.

Our study demonstrated relatively less apoptosis in theliver compared to the kidney following BD as demon-strated by the Bcl2/Bax. The comparatively low levels ofTNF-α in the liver following BD may in part explain this.However, prednisolone failed to have a further effect onreducing the Bcl2/Bax ratio.

In contrast to the kidney, HSP70 expression in theliver following BD and prednisolone pre-treatment wasdown-regulated. Stoot et al. [48] showed that HSP70 activ-ity could prevent C3 expression. Interestingly, our studydemonstrated an increase in C3 expression in the liverfollowing BD and prednisolone pre-treatment. It is likelythat prednisolone is reducing HSP70 expression in theliver but not in the kidney and thereby increase or facili-tate C3 deposition in the liver. This may explain the lackof prednisolone effect on reducing liver injury.

ConclusionThere are a number of differences in the way organsrespond to BD and donor treatment. We have demon-strated that the administration of prednisolone as apre-treatment agent to the BD donor is likely to havedifferential effects on the kidney and the liver. The lackof effect of prednisolone in our experimental set up couldin part, be due to the complement mediated injury. Weacknowledge that we have not evaluated the effect ofprednisolone after BD induction or time-course effect ofprednisolone therapy at earlier or later time-points dur-ing the BD period. We also have not evaluated the effectof compounding the injury during the donor period withorgan preservation and reperfusion injury or assessed theeffects of prednisolone. Nevertheless this study demon-strates the clear need to consider the differential effect ofdonor therapies on different organ systems and the needfor further research.

AbbreviationsADH: Antidiuretic hormone; LDH: Lactate dehydrogenase; T3:Triiodothyronine; T4: Thyroxine; AST: Aspartate aminotrasferase; ALT: Alanineaminotransferase; ACTH: Adrenocorticotropic hormone; ICAM-1: IntercellularAdhesion Molecule 1; VEGF: Vascular endothelial growth factor; vWF: VonWillebrand factor; IL-6: Interleukin 6; IL-10: Interleukin 10; TNF-α: Tumornecrosis factor; TGF-β : Transforming growth factor beta; MCP-1: Monocytechemotactic protein-1; NF-κB: Nuclear factor kappa-light-chain-enhancer ofactivated B cells; HAES: Hydroxyethyl starch; NA: Noradrenaline; GAPDH:Glyceraldehyde 3-phosphate dehydrogenase; C3: Complement component 3;Bcl2: B-cell lymphoma 2; BAX: Bcl-2 associated X protein; HO-1: Hemeoxygenase 1; PMNs: Polymorphonuclear neutrophils; IL-1β : Interleukin 1 beta.

Rebolledo et al. Journal of Translational Medicine 2014, 12:111 Page 9 of 10http://www.translational-medicine.com/content/12/1/111

Competing interestsThe authors declare no conflict of interests.

Authors’ contributionsRR. and LB. participated in research design, performance, data analysis and inthe writing of the paper. MZA. participated in the writing of the paper. PJO.participated in the performance of the research. JNZ and RJP participated inthe research design. HGL. participated in research design and in the writing ofthe paper. All authors read and approved the final manuscript.

AcknowledgementsWe would like to thank Dr. Pamela Romanque, assistant professor of Medicinein the University of Chile for her advice and careful revision during thepreparation of this manuscript.

Author details1Department of Surgery, University Medical Center Groningen, Hanzeplein 1,9713 GZ Groningen, The Netherlands. 2Department of Surgery, Faculty ofMedicine, University of Chile, Independencia 1027, 8380453 Santiago, Chile.3Department of Neurosurgery, Tianjin Medical University General Hospital, 154Anshan Road, 300052 Tianjin, China. 4Nuffield Department of SurgicalSciences, University of Oxford, Headington, OX3 9DU Oxford, UK.

Received: 13 February 2014 Accepted: 25 April 2014Published: 2 May 2014

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doi:10.1186/1479-5876-12-111Cite this article as: Rebolledo et al.: Prednisolone has a positive effect onthe kidney but not on the liver of brain dead rats: a potencial role incomplement activation. Journal of Translational Medicine 2014 12:111.

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